Structure of the Ni Sites in Hydrogenases by X-ray Absorption Spectroscopy. Species Variation and the Effects of Redox Poise

Abstract

Structural information obtained from the analysis of nickel K-edge X-ray absorption spectroscopic data of [NiFe]hydrogenases from Desulfovibrio gigas, Thiocapsa roseopersicina, Desulfovibrio desulfuricans (ATCC 27774), Escherichia coli (hydrogenase-1), Chromatium vinosum, and Alcaligenes eutrophus H16 (NAD+-reducing, soluble hydrogenase), poised in different redox states, is reported. The data allow the active-site structures of enzymes from several species to be compared, and allow the effects of redox poise on the structure of the nickel sites to be examined. In addition, the structure of the nickel site obtained from recent crystallographic studies of the D. gigas enzyme (Volbeda, A.; Charon, M.-H.; Piras, C.; Hatchikian, E. C.; Frey, M.; Fontecilla-Camps, J. C. Nature 1995, 373, 580−587) is compared with the structural features obtained from the analysis of XAS data from the same enzyme. The nickel sites of all but the oxidized (as isolated) sample of A. eutrophus hydrogenase are quite similar. The nickel K-edge energies shift 0.9−1.5 eV to lower energy upon reduction from oxidized (forms A and B) to fully reduced forms. This value is comparable with no more than a one-electron metal-centered oxidation state change. With the exception of T. roseopersicina hydrogenase, most of the edge energy shift (∼0.8 eV) occurs upon reduction of the oxidized enzymes to the EPR-silent intermediate redox level (SI). Analysis of the XANES features assigned to 1s → 3d electronic transitions indicates that the shift in energy that occurs for reduction of the enzymes to the SI level may be attributed at least in part to an increase in the coordination number from five to six. The smallest edge energy shift is observed for the T. roseopersicina enzyme, where the XANES data indicate that the nickel center is always six-coordinate. With the exception of the oxidized sample of A. eutrophus hydrogenase, the EXAFS data are dominated by scattering from S-donor ligands at ∼2.2 Å. The enzyme obtained from T. roseopersicina also shows evidence for the presence of O,N-donor ligands. The data from A. eutrophus hydrogenase are unique in that they indicate that a significant structural change occurs upon reduction of the enzyme. EXAFS data obtained from the oxidized (as isolated) A. eutrophus enzyme indicate that the EXAFS is dominated by scattering from 3−4 N,O-donor atoms at 2.06(2) Å, with contributions from 2−3 S-donor ligands at 2.35(2) Å. This changes upon reduction to a more typical nickel site composed of ∼4 S-donor ligands at a Ni−S distance of 2.19(2) Å. Evidence for the presence of atoms in the 2.4−2.9 Å distance range is found in most samples, particularly the reduced enzymes (SI, form C, and R). The analysis of these data is complicated by the fact that it is difficult to distinguish between S and Fe scattering atoms at this distance, and by the potential presence of both S and another metal atom at similar distances. The results of EXAFS analysis are shown to be in general agreement with the published crystal structure of th...

Spectroscopic and model studies of the Ni-Fe hydrogenase reaction mechanism

“…The Ni-Fe distance was determined to be 2.9 , and a large difference in the Ni-S distances between terminal (~2.2 ) and bridging (~2.6 ) cysteinate ligands was noted. However, the large difference in the Ni-S distances is not consistent with EXAFS analysis [11,25] and is not observed in the crystal structures of several dithiolato-bridged dinickel complexes [15,26,27].…”

“…X-ray absorption data have been used to compare structures of Ni-Fe active sites in enzymes obtained from different species and to examine structural changes that accompany redox chemistry, photochemistry, and inhibitor (CO) binding [10,11,25]. In general, the structures of the Ni sites in reduced Ni-Fe H 2 ases are quite similar, but the structures of oxidized enzymes show some variability.…”

The Auxiliary Protein HypX Provides Oxygen Tolerance to the Soluble [NiFe]-Hydrogenase of Ralstonia eutropha H16 by Way of a Cyanide Ligand to Nickel

“…They pointed to the presence of more hard ligands (N, O) to nickel in the SH than found in standard hydrogenases. Hence, it has been proposed that one or more of the conserved Cys thiols in the HoxH subunit may not be directly coordinated to nickel (27,28). 3 Biosynthesis of the unique Ni-Fe active site is a proteinassisted process.…”

Intermediates in the Catalytic Cycle of [NiFe] Hydrogenase: Functional Spectroscopy of the Active Site

“…[24,25] The combination with electrochemical techniques allows the pH-dependent determination of the redox transitions. Together with quantum-chemical calculations these and other techniques, like Mçssbauer [34] or XAS [35,36] spectroscopies, are helpful in elucidating the details of the reaction mechanism for the [NiFe] hydrogenases. [32,[37][38][39] The aim of this minireview is to give an overview of the current knowledge of this metalloenzyme, relate it to other species and outline the advantages and problems for the potential use of hydrogenases in future energy technologies.…”